The present invention relates to methods and devices employing a colored dye compound covalently bound to a supporting matrix. More particularly, the colored dye compound is formed by the coupling of a first and second component of a two component dye system, the second component of which is covalently immobilized on the matrix. Addition of the first dye component forms a colored covalent adduct covalently immobilized to the matrix. The methods of making, the methods of using and the devices themselves have particular utility in test strips used in diagnostic medicine wherein the colored dye compounds are permanently affixed to the matrix and do not run or leach.
A number of methods binding a dye to a matrix are known in the art. The textile industry binds dye to textiles using mordants which, acting alone or in conjunction with a dye, become absorbed or absorbed or otherwise intercalate and become stuck on the surface or on the fibers of the textile. Because the mordants and dyes are not covalently bound to the textile, they tend to leach out with washing causing fading and discoloration.
Other methods for immobilizing dye compounds include providing a dye molecule with a higher alkyl hydrophobic side chain which inserts itself into a hydrophobic substrate or support and is immobilized by hydrophilic/hydrophobic interactions. See, for example, Bloom, et al., U.S. Pat. No. 3,443,939.
Nevertheless, colored dye compounds used in analytical test devices have been bound only unsatisfactorily to date. Typically, a colored compound is immobilized on the basis of its insolubility relative to the assay solution, which causes it to precipitate onto the matrix without covalent bonding. In other systems, generated color is absorbed, imbibed, impregnated, or coated onto the supporting matrix. Patents exemplifying this approach include U.S. Pat. Nos. 4,069,016, 4,548,905, 4,038,031. Finally, limited success at immobilizing color has been achieved by generating localized, precipitated color only at the surface of the solid phase through a signal generating enzyme system immobilized to the support. See, for example, U.S. Pat. No. 4,435,504.
It is an object of the present invention to overcome the, disadvantages of the prior art and to provide methods and apparatus in which the colored indicating compound is covalently immobilized to the support and cannot leach or be washed therefrom.